Viral shedding and viraemia of Andes virus during acute hantavirus infection: a prospective study.

BACKGROUND: Andes virus (ANDV) is a zoonotic Orthohantavirus leading to hantavirus cardiopulmonary syndrome. Although most transmissions occur through environmental exposure to rodent faeces and urine, rare person-to-person transmission has been documented, mainly for close contacts. This study investigates the presence and infectivity of ANDV in body fluids from confirmed cases and the duration of viraemia. METHODS: In this prospective study, 131 participants with confirmed ANDV infection were enrolled in Chile in a prospective study between 2008 and 2022. Clinical samples (buffy coat, plasma, gingival crevicular fluid [GCF], saliva, nasopharyngeal swabs [NPS], and urine) were collected weekly for 3 weeks together with clinical and epidemiological data. Samples were categorised as acute or convalescent (up to and after 16 days following onset of symptoms). Infectivity of positive fluids was assessed after the culture of samples on Vero E6 cells and use of flow cytometry assays to determine the production of ANDV nucleoprotein. FINDINGS: ANDV RNA was detected in 100% of buffy coats during acute phase, declining to 95% by day 17, and to 93% between days 23-29. ANDV RNA in GCF and saliva decreased from 30% and 12%, respectively, during the acute phase, to 12% and 11% during the convalescent phase. Successful infectivity assays of RT-qPCR-positive fluids, including GCF, saliva, NPS, and urine, were observed in 18 (42%) of 43 samples obtained during the acute phase of infection. After re-culture, the capacity to infect Vero E6 cells was maintained in 16 (89%) of 18 samples. Severity was associated with the presence of ANDV RNA in one or more fluids besides blood (odds ratio 2·58 [95% CI 1·42-5·18]). INTERPRETATION: ANDV infection is a systemic and viraemic infection, that affects various organs. The presence of infectious particles in body fluids contributes to our understanding of potential mechanisms for person-to-person transmission, supporting the development of preventive strategies. Detection of ANDV RNA in additional fluids at hospital admission is a predictor of disease severity. FUNDING: National Institutes of Health and Agencia de Investigación y Desarrollo. TRANSLATION: For the Spanish translation of the abstract see Supplementary Materials section.

Hypervirulent Strains of Neisseria meningitidis and Clinical Manifestations in Children With Invasive Meningococcal Disease.

BACKGROUND: Hypervirulent clonal complex (cc) have been associated with higher incidence and case fatality rate of invasive meningococcal disease (IMD). The aim of this study was to describe the clinical manifestations of the hypervirulent cc of meningococcus in children. METHODS: Retrospective study in patients hospitalized by IMD microbiologically confirmed at three children's tertiary health care centers in Santiago, Chile, between 2010 and 2018. Demographic, clinical information and determination of the cc and factor H binding protein (fHbp) alleles were performed. RESULTS: In total 93 cases were evaluated, sequence typing was available for 91 cases, and 87 (95.6%) had a cc assigned; 63.7% were MenW and 31.8% MenB. The median age was 9 months, 67% were male and 18.7% had any comorbidity. A 26.4% presented neurological deficit, 25.3% petechiae and 20% diarrhea. Sixty-seven percent were admitted to the pediatric intensive care unit (PICU) and the case fatality rate was 9.9%. Regarding cc and fHbp alleles, ST11, ST41/44 and allele 22 were the most frequently identified, with 63.7%, 19.8% and 72.5%, respectively. We found statistically significant differences between the cc and presence of petechiae, diagnosis of meningococcemia plus meningitis, admission and days in PICU and advanced support. Allele 22 for fHbp was associated with the absence of petechiae, low suspicion of IMD, less diagnosis of meningitis+meningococcemia, PICU admission, advanced support and adrenal insufficiency. CONCLUSION: Epidemiological and microbiological surveillance of IMD should integrate clinical and laboratory components, including molecular and genetic characterization, to enrich the dynamic understanding of the clinical evolution of IMD.

Broad and potently neutralizing monoclonal antibodies isolated from human survivors of New World hantavirus infection.

New World hantaviruses (NWHs) are endemic in North and South America and cause hantavirus cardiopulmonary syndrome (HCPS), with a case fatality rate of up to 40%. Knowledge of the natural humoral immune response to NWH infection is limited. Here, we describe human monoclonal antibodies (mAbs) isolated from individuals previously infected with Sin Nombre virus (SNV) or Andes virus (ANDV). Most SNV-reactive antibodies show broad recognition and cross-neutralization of both New and Old World hantaviruses, while many ANDV-reactive antibodies show activity for ANDV only. mAbs ANDV-44 and SNV-53 compete for binding to a distinct site on the ANDV surface glycoprotein and show potently neutralizing activity to New and Old World hantaviruses. Four mAbs show therapeutic efficacy at clinically relevant doses in hamsters. These studies reveal a convergent and potently neutralizing human antibody response to NWHs and suggest therapeutic potential for human mAbs against HCPS.

[Molecular detection of gastrointestinal pathogens among children under 5 years old with diarrhea in a hospital center for rotavirus sentinel surveillance in Chile]. / Detección molecular de patógenos entéricos en niños con diarrea en un hospital centinela de vigilancia de rotavirus en Chile.

BACKGROUND: Infectious diarrhea is still a major problem in public health, especially in children under 5 years of age. The identification of the etiologic agent is important for the clinical management of the diarrhea episode and, from the epidemiological point of view, to implement control measures. AIM: To determine the presence of gastrointestinal pathogens in children under five years of age with diarrhea in a Chilean rotavirus surveillance center. METHODS: Observational study in children under five years of age who were hospitalized for diarrhea at the Dr. Luis Calvo Mackenna Hospital from December 2015 to December 2019. Molecular detection was performed using the FilmArray gastrointestinal (FilmArray GI®) panel. RESULTS: We analyzed 493 diarrheal stool samples of children, 427 samples (87%) were positive and 66 samples (13%) were negative. Of positive samples, 174 samples (41%) and 253 samples (59%) were positive for one or more pathogen, respectively. In children under one year and the group between one and four years there was a predominance of infections caused by enteric virus. Rotavirus and norovirus were the most common virus in both age groups. The most frequent bacteria were EPEC (27%), C. difficile (17%), EAEC (14%) and Campylobacter (9%). In parasites, Giardia lamblia and Cryptosporidium were identified, in 3% and 1% of the total samples, respectively. CONCLUSIONS: The molecular detection system used allowed an increase in the detection of enteropathogens in children under five years of age. The information generated by this type of surveillance could help to characterize the episodes of diarrhea in the population and might be a tool to technically advise the authorities in the decision-making process for the implementation of control measures.

Detección molecular de patógenos entéricos en niños con diarrea en un hospital centinela de vigilancia de rotavirus en Chile / Molecular detection of gastrointestinal pathogens among children under 5 years old with diarrhea in a hospital center for rotavirus sentinel surveillance in Chile

INTRODUCCIÓN: Las diarreas de causa infecciosa son un problema de salud pública, especialmente en niños bajo los cinco años. La identificación de los agentes etiológicos puede ser relevante para el manejo del cuadro clínico y, desde el punto de vista epidemiológico, para la implementación de medidas de control. OBJETIVO: Determinar la presencia de patógenos entéricos en niños bajo los cinco años que se hospitalizaron por diarrea aguda en uno de los centros centinelas de la red de vigilancia de rotavirus en Chile. PACIENTES Y MÉTODOS: Estudio observacional en niños menores de cinco años que se internaron por cuadros de diarrea en el Hospital Dr. Luis Calvo Mackenna, durante diciembre del 2015 a diciembre del 2019, el que forma parte de la red de vigilancia de rotavirus del Ministerio de Salud de Chile. Las muestras fecales se analizaron mediante un test molecular, FilmArray GI® panel, que permite la detección de 22 patógenos entéricos virales, bacterianos y parasitarios. RESULTADOS: Se analizaron 493 muestras fecales de niños con episodios de diarrea infecciosa, detectando al menos un patógeno en 427 muestras (87%). De estas muestras positivas, se detectó solo un patógeno en 174 muestras (41%) y dos o más patógenos en 253 muestras (59%). En el grupo de niños bajo un año y el grupo entre uno y cuatro años hubo un predominio de infecciones causadas por virus gastroentéricos, siendo rotavirus y norovirus los virus más detectados en ambos grupos de edad. Las bacterias más frecuentes fueron EPEC (27%), C. difficile (17%), EAEC (14%) y Campylobacter (9%). Respecto a los parásitos, se identificó Giardia lamblia y Cryptosporidium, en el 3 y 1% del total de las muestras, respectivamente. CONCLUSIÓN: La detección molecular utilizada permitió detectar un alto número de enteropatógenos en niños bajo los cinco años. La información generada por este tipo de vigilancia, podría ayudar a caracterizar en la población los episodios de diarrea causados por los principales patógenos entéricos y podría ser una herramienta para asesorar técnicamente a las autoridades en la toma de decisión para la implementación de medidas de control contra estos patógenos.

BACKGROUND: Infectious diarrhea is still a major problem in public health, especially in children under 5 years of age. The identification of the etiologic agent is important for the clinical management of the diarrhea episode and, from the epidemiological point of view, to implement control measures. AIM: To determine the presence of gastrointestinal pathogens in children under five years of age with diarrhea in a Chilean rotavirus surveillance center. METHODS: Observational study in children under five years of age who were hospitalized for diarrhea at the Dr. Luis Calvo Mackenna Hospital from December 2015 to December 2019. Molecular detection was performed using the FilmArray gastrointestinal (FilmArray GI®) panel. RESULTS: We analyzed 493 diarrheal stool samples of children, 427 samples (87%) were positive and 66 samples (13%) were negative. Of positive samples, 174 samples (41%) and 253 samples (59%) were positive for one or more pathogen, respectively. In children under one year and the group between one and four years there was a predominance of infections caused by enteric virus. Rotavirus and norovirus were the most common virus in both age groups. The most frequent bacteria were EPEC (27%), C. difficile (17%), EAEC (14%) and Campylobacter (9%). In parasites, Giardia lamblia and Cryptosporidium were identified, in 3% and 1% of the total samples, respectively. CONCLUSIONS: The molecular detection system used allowed an increase in the detection of enteropathogens in children under five years of age. The information generated by this type of surveillance could help to characterize the episodes of diarrhea in the population and might be a tool to technically advise the authorities in the decision-making process for the implementation of control measures.

Comparison of VSV Pseudovirus and Focus Reduction Neutralization Assays for Measurement of Anti-Andes orthohantavirus Neutralizing Antibodies in Patient Samples.

Andes orthohantavirus (ANDV) is the etiologic agent of hantavirus cardiopulmonary syndrome (HCPS), which has a case fatality rate around 35%, with no effective treatment or vaccine available. ANDV neutralizing antibody (NAb) measurements are important for the evaluation of the immune response following infection, vaccination, or passive administration of investigational monoclonal or polyclonal antibodies. The standard assay for NAb measurement is a focus reduction neutralization test (FRNT) featuring live ANDV and must be completed under biosafety level (BSL)-3 conditions. In this study, we compared neutralization assays featuring infectious ANDV or vesicular stomatitis virus (VSV) pseudovirions decorated with ANDV glycoproteins for their ability to measure anti-ANDV NAbs from patient samples. Our studies demonstrate that VSV pseudovirions effectively measure NAb from clinical samples and have greater sensitivity compared to FRNT with live ANDV. Importantly, the pseudovirus assay requires less labor and sample materials and can be conducted at BSL-2.

A Multicenter Study To Evaluate Ceftaroline Breakpoints: Performance in an Area with High Prevalence of Methicillin-Resistant Staphylococcus aureus Sequence Type 5 Lineage.

Ceftaroline (CPT) is a broad-spectrum agent with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). The sequence type 5 (ST5) Chilean-Cordobés clone, associated with CPT nonsusceptibility, is dominant in Chile, a region with high rates of MRSA infections. Here, we assessed the in vitro activity of CPT against a collection of MRSA isolates collected between 1999 and 2018 from nine hospitals (n = 320) and community settings (n = 41) in Santiago, Chile, and evaluated performance across testing methodologies. We found that our hospital-associated isolates exhibited higher CPT MIC distributions (MIC50 and MIC90 of 2 mg/liter) than the community isolates (MIC50 and MIC90 of 0.5 mg/liter), a finding that was consistent across time and independent of the culture source. High proportions (64%) of isolates were CPT nonsusceptible despite the absence of CPT use in Chile. Across methodologies, the Etest underestimated the MIC relative to the gold standard broth microdilution (BMD) test (MIC50 and MIC90 of 1 and 1.5 mg/liter, respectively). There was low (∼51%) categorical agreement (CA) between Etest and BMD results across CLSI and EUCAST breakpoints. The recent revision of CLSI guidelines abolished "very major error" (VME) from the previous guidelines (81%), which perform similarly to the EUCAST guidelines. The level of concordance between CLSI and EUCAST for BMD testing and Etest was >95%. Disk diffusion performed poorly relative to BMD under CLSI (CA, 55%) and EUCAST (CA, 36%) guidelines. Comparison of EUCAST to CLSI for disk diffusion (with EUCAST used as the reference) showed low agreement (CA, 25%; VME, 70%). In summary, CPT-nonsusceptible MRSA are dominant in clinical settings in Chile. Our results provide data to support the reevaluation of CPT breakpoints and to improve agreement across methodologies and agencies.

Yellow fever vaccination for Chilean tourists visiting Brazil. Practical considerations. / Vacuna contra fiebre amarilla para viajeros chilenos a Brasil. Consideraciones prácticas.

Due to the recent yellow fever outbreak affecting the costal region of Brazil, including main touristic destinations, there is a high demand of yellow fever vaccination. This publication addresses the most relevant practical issues regarding this vaccine for tourists visiting Brazil and aims to serve as a guideline for non-expert physicians in Chile and elsewhere.

Vacuna contra fiebre amarilla para viajeros chilenos a Brasil. Consideraciones prácticas / Yellow fever vaccination for Chilean tourists visiting Brazil. Practical considerations

Resumen La aparición de fiebre amarilla en las costas brasileñas, lugares de alto interés turístico, ha provocado una alta demanda de vacunación. Este articulo entrega respuestas a las principales consultas sobre la vacuna de fiebre amarilla y puede servir como guía para médicos no expertos en Medicina del Viajero.

Due to the recent yellow fever outbreak affecting the costal region of Brazil, including main touristic destinations, there is a high demand of yellow fever vaccination. This publication addresses the most relevant practical issues regarding this vaccine for tourists visiting Brazil and aims to serve as a guideline for non-expert physicians in Chile and elsewhere.

Knowledge, attitudes, and practices regarding hantavirus disease and acceptance of a vaccine trial in rural communities of southern Chile.

Andes hantavirus cardiopulmonary syndrome, transmitted by Oligoryzomys longicaudatus, has no approved treatment, a case fatality rate of 35%, and documented person-to-person transmission. An Andes vaccine, highly needed for prevention, is in development. We aimed to evaluate knowledge, attitudes and practices (KAP) regarding hantavirus disease and willingness to participate in a future Andes vaccine trials through a cross sectional face-to-face oral survey of a randomly selected adult sample from 2 rural communes in southern Chile. Human subjects approval was obtained from our institutional IRBs, and participants signed informed consent. We enrolled 319 subjects from Corral and 321 from Curarrehue; 98% had heard about hantavirus disease and its reservoir but only half knew about transmission, symptoms and prevention. Participants fear the disease but are only partially aware of their own risk. One third of participants reported presence of rodents inside their homes. Despite moderate confidence in their health system, most subjects perceived vaccines as beneficial, and 93% would accept an approved hantavirus vaccine. Half would agree to participate in a vaccine trial and 29% would allow their children to participate. Motivations to participate were mainly altruistic, while risk perception was the main reason for declining. Knowledge about hantavirus disease and prevention practices require reinforcement, and a vaccine trial seems feasible in these populations.

Molecular method for the detection of Andes hantavirus infection: validation for clinical diagnostics.

Hantavirus cardiopulmonary syndrome is a severe disease caused by exposure to New World hantaviruses. Early diagnosis is difficult due to the lack of specific initial symptoms. Antihantavirus antibodies are usually negative until late in the febrile prodrome or the beginning of cardiopulmonary phase, while Andes hantavirus (ANDV) RNA genome can be detected before symptoms onset. We analyzed the effectiveness of quantitative reverse transcription polymerase chain reaction (RT-qPCR) as a diagnostic tool detecting ANDV-Sout genome in peripheral blood cells from 78 confirmed hantavirus patients and 166 negative controls. Our results indicate that RT-qPCR had a low detection limit (~10 copies), with a specificity of 100% and a sensitivity of 94.9%. This suggests the potential for establishing RT-qPCR as the assay of choice for early diagnosis, promoting early effective care of patients, and improving other important aspects of ANDV infection management, such as compliance of biosafety recommendations for health personnel in order to avoid nosocomial transmission.

Hantavirus pulmonary syndrome, Southern Chile, 1995-2012.

Hantavirus is endemic to the Region de Los Lagos in southern Chile; its incidence is 8.5 times higher in the communes of the Andean area than in the rest of the region. We analyzed the epidemiologic aspects of the 103 cases diagnosed by serology and the clinical aspects of 80 hospitalized patients during 1995-2012. Cases in this region clearly predominated during winter, whereas in the rest of the country, they occur mostly during summer. Mild, moderate, and severe disease was observed, and the case-fatality rate was 32%. Shock caused death in 75% of those cases; high respiratory frequency and elevated creatinine plasma level were independent factors associated with death. Early clinical suspicion, especially in rural areas, should prompt urgent transfer to a hospital with an intensive care unit and might help decrease the high case-fatality rate.

A non-randomized multicentre trial of human immune plasma for treatment of hantavirus cardiopulmonary syndrome caused by Andes virus.

BACKGROUND: In Chile, Andes virus (ANDV) is the sole aetiological agent of hantavirus cardiopulmonary syndrome (HCPS) with mean annual incidence of 55 cases, 32% case fatality rate (CFR) and no specific treatment. Neutralizing antibody (NAb) titres at hospital admission correlate inversely with HCPS severity. We designed an open trial to explore safety and efficacy and evaluate pharmacokinetics of immune plasma as a treatment strategy for this disease. METHODS: We performed plasmapheresis on donors at least 6 months after HCPS and measured NAb titres through a focus-reduction neutralization test. Subjects admitted to 10 study sites with suspected/confirmed HCPS were eligible for treatment with immune plasma by intravenous infusion at an ANDV NAb dose of 5,000 U/kg. HCPS was confirmed through immunoglobulin M serology or reverse transcriptase-PCR. The main outcome was mortality within 30 days. RESULTS: From 2008-2012, we enrolled and treated 32 cases and confirmed HCPS in 29. CFR of hantavirus plasma-treated cases was 4/29 (14%); CFR of non-treated cases in the same period in Chile was 63/199 (32%; P=0.049, OR=0.35, CI=0.12, 0.99); CFR of non-treated cases at the same study sites between 2005-2012 was 18/66 (27%; (P=0.15, OR=0.43, CI=0.14, 1.34) and CFR in a previous methylprednisolone treatment study was 20/60 (33%; P=0.052, OR=0.32, CI=0.10, 1.00). We detected no serious adverse events associated to plasma infusion. Plasma NAb titres reached in recipients were variable and viral load remained stable. CONCLUSIONS: Human ANDV immune plasma infusion appears safe for HCPS. We observed a decrease in CFR in treated cases with borderline significance that will require further studies for confirmation.

Person-to-person household and nosocomial transmission of andes hantavirus, Southern Chile, 2011.

Andes hantavirus (ANDV) causes hantavirus cardiopulmonary syndrome in Chile and is the only hantavirus for which person-to-person transmission has been proven. We describe an outbreak of 5 human cases of ANDV infection in which symptoms developed in 2 household contacts and 2 health care workers after exposure to the index case-patient. Results of an epidemiologic investigation and sequence analysis of the virus isolates support person-to-person transmission of ANDV for the 4 secondary case-patients, including nosocomial transmission for the 2 health care workers. Health care personnel who have direct contact with ANDV case-patients or their body fluids should take precautions to prevent transmission of the virus. In addition, because the incubation period of ANDV after environmental exposure is longer than that for person-to-person exposure, all persons exposed to a confirmed ANDV case-patient or with possible environmental exposure to the virus should be monitored for 42 days for clinical symptoms.

High-dose intravenous methylprednisolone for hantavirus cardiopulmonary syndrome in Chile: a double-blind, randomized controlled clinical trial.

BACKGROUND: Andes virus (ANDV)-related hantavirus cardiopulmonary syndrome (HCPS) has a 35% case fatality rate in Chile and no specific treatment. In an immunomodulatory approach, we evaluated the efficacy of intravenous methylprednisolone for HCPS treatment, through a parallel-group, placebo-controlled clinical trial. METHODS: Patients aged >2 years, with confirmed or suspected HCPS in cardiopulmonary stage, admitted to any of 13 study sites in Chile, were randomized by study center in blocks of 4 with a 1:1 allocation and assigned through sequentially numbered envelopes to receive placebo or methylprednisolone 16 mg/kg/day (≤1000 mg) for 3 days. All personnel remained blinded except the local pharmacist. Infection was confirmed by immunoglobulin M antibodies or ANDV RNA in blood. The composite primary endpoint was death, partial pressure of arterial oxygen/fraction of inspired oxygen ratio ≤55, cardiac index ≤2.2, or ventricular tachycardia or fibrillation within 28 days. Safety endpoints included the number of serious adverse events (SAEs) and quantification of viral RNA in blood. Analysis was by intention to treat. RESULTS: Infection was confirmed in 60 of 66 (91%) enrollees. Fifteen of 30 placebo-treated patients and 11 of 30 methylprednisolone-treated patients progressed to the primary endpoint (P = .43). We observed no significant difference in mortality between treatment groups (P = .41). There was a trend toward more severe disease in placebo recipients at entry. More subjects in the placebo group experienced SAEs (P = .02). There were no SAEs clearly related to methylprednisolone administration, and methylprednisolone did not increase viral load. CONCLUSIONS: Although methylprednisolone appears to be safe, it did not provide significant clinical benefit to patients. Our results do not support the use of methylprednisolone for HCPS. CLINICAL TRIALS REGISTRATION: NCT00128180.

Highly differentiated, resting gn-specific memory CD8+ T cells persist years after infection by andes hantavirus.

In man, infection with South American Andes virus (ANDV) causes hantavirus cardiopulmonary syndrome (HCPS). HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-gamma ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3(+)CD8(+) T cells were specific for the single HLA-B*3501-restricted epitope Gn(465-473) years after the acute infection. Remarkably, Gn(465-473)-specific cells readily secreted IFN-gamma, granzyme B and TNF-alpha but not IL-2 upon stimulation and showed a 'revertant' CD45RA(+)CD27(-)CD28(-)CCR7(-)CD127(-) effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T cells are critical for viral control and protective immunity. If so, Gn-derived immunodominant epitopes could be of high value for future ANDV vaccines.

Case report: T-cell responses during clearance of Andes virus from blood cells 2 months after severe hantavirus cardiopulmonary syndrome.

Hantavirus Cardiopulmonary Syndrome (HCPS) due to Andes virus (ANDV) is endemic in Chile and Argentina and currently demonstrates a case-fatality rate of 37% in humans. By contrast to the chronically infected rodents, it is believed that ANDV in humans is cleared during the acute phase. Moreover, to date, both magnitude and quality of human T-cell responses during ANDV infection and clearance are unknown. Using IFN-gamma and granzyme B ELISPOT assays as well as flow cytometry, we prospectively studied the ANDV-specific T-cell responses in a 56-year-old convalescing survivor of severe HCPS, whose blood cells remained PCR-positive for ANDV-RNA until day 53 after hospital admission, that is, 67 days after infection and 42 days after discharge. PCR-negativity was closely related to the increase and function of (Gn(46-60))-specific IFN-gamma(+) granzyme B(+) CD8(+) T-cells, but not to neutralizing antibody titers. Concurrently, the phenotype of CD45RA(+)CCR7(-) Gn(46-60)-specific T-cells shifted from a CD28(-)CD27(+) "intermediate" to a CD28(-) CD27(-) "late" effector memory beyond day 53 after hospital admission. This is the first report that shows that ANDV can persist in the human hosts for more than 2 months. Moreover, the kinetics of T-cell responses during ANDV clearance may indicate a major role of T-cells for clearance of ANDV and human immunity to this pathogen.

Incubation period of hantavirus cardiopulmonary syndrome.

The potential incubation period from exposure to onset of symptoms was 7-39 days (median 18 days) in 20 patients with a defined period of exposure to Andes virus in a high-risk area. This period was 14-32 days (median 18 days) in 11 patients with exposure for <48 hours.